Genome-wide mutagenesis in lower organisms (e.g. bacteria, nematodes, yeast, zebra fish and Drosophila) followed by screening or selection for mutants using phenotypic assays has proven to be a useful methodology for revealing gene function in these organisms.
The mouse provides a very useful mammalian animal model for studying gene function. The mouse model possesses significant advantages because of its evolutionary relatedness to humans, similarity to humans with respect to the development of complex tissues and organs, and because it provides opportunity to rapidly identify homologous genes through regions of genomic sestina. Large-scale mutagenesis in programs using mice now play a significant role in the study of mammalian gene function (Brown & Nolan (1998) Human Molecular Genetics, 7:1627-1633). The mutagen of choice for use in large-scale mouse studies is N-ethyl-N-nitrosourea (ENU) which is administered to male mice.
Technological advances in culture and maintenance of embryonic stem (ES) cells has provided new opportunities for study of eukaryotic genomes including that of the mouse. Murine ES cells are derived from the inner cell mass of about a 3.5 day embryo or blastocyst and can be maintained in an undifferentiated, pluripotent state in culture. ES cells can be genetically manipulated in vitro and these cells may subsequently be introduced into an embryo by blastocyst microinjection or embryo aggregation techniques. Upon reintroduction into the embryo, ES cells can contribute to the formation of all tissues of the resulting chimeric organism. ES cell contribution to germ cells of the reproductive organs results in germline transmission of mutations introduced into the ES cell genome. For these reasons, mutation of ES cells is used as another means for generating mutations in the mouse genome. For example, murine ES cells may be irradiated (Brown & Nolan [supra]) or mutated through the use of insertional mutagenesis such as transposon tagging, retroviral integration, or gene trap mutagenesis.
Screening strategies in mouse mutagenesis programs vary according to phenotype under study and according to the means by which mutations are produced. For example, various expression based strategies are described for screening cell lines or animals derived from ES cells in which a gene trap vector has been used to generate a mutation (e.g. Baker, et al. (1997) Dev. Biol., 185:201-14; Kuwano, R. (1996) Zool. Sci., 13:277-83; Wurst, et al. (1995) Genetics, 139:889-99; and PCT application published Jan. 21, 1999 under WO 99/02719). While the above-described methodologies which make use of large-scale mutagenesis are used for study of the murine genome, gene sequence based systems have also been developed and are concurrently used for analysis of the mouse genome. The latter approach is expected to be used in parallel with mutagenic approaches to provide an enlarged catalogue of mouse mutations and phenotypes for gene function studies (Brown & Nolan [supra]).
The current gene sequence based strategy of choice for the mouse makes use of the production of a library of ES gene trap clones indexed by either polynucleotide fragments derived from regions flanking the site of gene trap integration or by DNA sequence information derived from such fragments. The premise behind this approach is that most mammalian genes will soon be characterized from sequences of “expressed sequence tags” (ESTs). An example of such an ES cell library is known as Omnibank™ and is described, for example by Brown & Nolan [supra], Zambrowicz, et al. (1998) Nature, 392:608-611, and in U.S. Pat. Nos. 6,136,566 and 6,207,371. Another example is described in Wiles, M. V. et al. (2000) Nature Genetics, 24:13-14. Such libraries may be generated by introducing an exon trap vector into ES cells and cloning separate cell lines representing individual trap vector integration events. The exon trap vector described by Zambrowicz, et al. (e.g. construct VICTR 20) comprises an upstream mutagenic cassette containing a splice acceptor (SA) sequence fused to a selectable reporter gene followed by a polyadnylation (polyA) sequence. This portion of the vector interrupts expression of the endogenous gene. A downstream portion of the trap vector ensures that integration of the trap into an exon may be detected without transcription of the endogenous gene. This downstream portion contains a promoter functional in the ES cell, linked to a reporter gene followed by a splice donor (SD) sequence. The promoter drives expression of the reporter gene together with endogenous DNA downstream to an endogenous polyA site. Sequence tags from endogenous (trapped) genes may be readily recovered using 3′ RACE-PCR, which generates polynucleotides corresponding to the regions which flank the site of integration of the vector. Furthermore, disruption of the endogenous gene by an exon trap vector permits one to readily generate transgenic and “knock-out” mice which are heterozygous for the mutation or are entirely deficient in the trapped gene function. This is accomplished using the ES cell methodologies described above. Chimeric animals that are generated by this procedure may be bred to provide homologous mutants. Further information regarding the construction and use of exon trap vectors, amplification of flanking regions, and generation of chimeric animals is found in WO 99/02719.